Protection against otitis media, non-complicated pneumonia, and meningitis requires a vaccine solution that is not capsular type-specific. The pneumococcal surface protein PspA elicits protection in mice against pneumonia, bacteremia, colonization, and fatal sepsis. PspA was in FDA-approved safety (phase 1) trials (1998, 2002, 2010, and 2012-14). These trials revealed no safety problems, and the human antibody (Ab) elicited to PspA protected mice from fatal sepsis. The human trials of purified rPspA (1998, 2002, and 2010) examined protection elicited by the ~300 aa a-helical domain (aHD) at the N-terminal end of PspA. The aHD is variable; up to three different aHD may be required in a vaccine to cover the diversity of PspA in all Sp. We have recently shown that the ~80 aa proline-rich domain (PRD) also elicits protection against sepsis. The PRD domain is found in all PspAs. Each PRD contains 1 to 5 different 6-10 AA repeat sequences. We have protective monoclonal antibody (mAb) to one of these short repeat sequences and to a conserved 22 aa non-proline block (NBP) sequence, present in ~50% of PRD. However, a PRD lacking either of these epitopes could still elicit cross-protection, indicating the existence of additional protective epitopes. We have already found Ab to four of the PRD repeat sequences in human sera. We will create broadly cross-reactive rPRD by including all of its common protection-eliciting epitopes. Developing a highly cross-protective PRD immunogen is important because some of the delay in moving PspA into phase 3 efficacy trials is due to concern that variability in the aHD structure may allow Sp to evolve around a PspA aHD vaccine. The PRD will provide a second set of epitopes with which to cover all PspAs. We will determine the degree to which rPRD can replace, or augment, the cross-protection elicited by the aHD against Sp strains of all relevant capsular types. Aim1. Identify major protection-eliciting repeats in PRD that are immunogenic in mice, rabbits, and humans and use this information to construct candidate rPRD expected to be broadly cross-protective. Aim2. Evaluate cross- protection elicited by the rPRD immunogens constructed in Aim 1 in mouse sepsis, focal pneumonia, and colonization models. Compare the ability of PRD, aHD, and PRD+aHD to elicit protection against diverse Sp. Variations in PspA structure are not genetically or functionally related to capsule type. Thus, it has long been assumed that the protection observed against the few mouse-virulent capsule types with Ab to PspA would hold for Sp of non-mouse virulent capsular types. To examine protection against invasive strains of all relevant capsular types we will: 1) use our in vitro functional assay, 2) ue focal pneumonia which is caused in the mouse by many more capsular types than sepsis, and 3) use host inflammation-associated molecules to temporarily enhance the virulence of invasive clinical IPD isolates of capsular types that are not normally mouse-virulent. Aim3. Determine whether Ab to PRD can enhance C' deposition on Sp and enhance the killing of Sp by bactericidal peptides. Ab to the aHD of PspA functions by both of these mechanisms.